The culture process for mammalian cells, animal cells, insect cells, bacteria, yeast and molds has one major rate limiting step, oxygen mass transfer. Oxygen metabolism is essential for metabolic function. In mammalian and animal cell culture it is especially important during the early stages of rapid cell division. Oxygen utilization per cell is greatest when cells are suspended; requirements decreasing as the cells aggregate and differentiate. However, during the later phases of cell culture, as the number of cells per unit volume increases, the bulk oxygen mass transfer requirements increase. Traditionally, increased requirements are accommodated by mechanical stirring methods. The present invention both randomizes the hydrodynamic flow, disrupts the establishment of boundary layers, enlarges the gas transfer surface area, and enhances mass transfer while allowing 3-dimensional tissue growth.
There are several basic strategies for increasing the gas mass transfer across a membrane: increase oxygen concentration; increase the rate of transfer from the air to the media; and/or increase the surface area for gas exchange. Increasing the oxygen partial pressure will increase the bulk oxygen transfer. However, a boundary layer of oxygen toxicity will form at the gas permeable membrane-media interface. Cells entering the toxic boundary layer could sustain irreparable damage. Approaches to increasing the rate of gas transfer at the air-membrane-media interface include: increase the rate of air movement across the membrane with air pumps or other mechanical means; increase the gas diffusion rate across the membrane by selecting a more gas permeable membrane; and/or increase the rate of media flow past the membrane. In each approach the rate of exchange across the gas permeable membrane is augmented, leading to improved gas exchange. The third approach for increasing bulk gas transfer is to increase the air-membrane-media surface area. An enlarged surface enhances the bulk gas transfer. Improved gas exchange serves to both increase oxygen availability and remove the carbon dioxide by-product. Carbon dioxide has a higher diffusivity constant in silicone material, gas permeable silicone membranes will more readily remove carbon dioxide.
The majority of mammalian and animal cells require cell-to-cell contact and 3-dimensional growth to differentiate into tissue. Recent technology has been established to grow mammalian cells in 3-dimensional constructs using rigid horizontally rotating bio-reactors developed by NASA, with forced air oxygenation through precision milled and drilled shafts. Such devices, having rigid non gas permeable walls defining the cell culture chambers are disclosed in U.S. Pat. No. 5,026,650, entitled "Horizontally Rotated Cell Culture System with a Coaxial Tubular Oxygenator", and U.S. Pat. No. 5,153,131, entitled "High Aspect Ratio Vessel and Method of Use." They are not adaptable for use with existing laboratory equipment, and require specific air pumps and have gas transfer rates that are limited. U.S. Pat. No. 5,330,908, "High Density Cell Culture System", discloses a process to increase the oxygenating surface area using a cell culture chamber that is rigid but oxygen permeable. However, all of the above horizontally rotating cell culture vessels have rigid walls that define the cell culture chamber and the stated invention is the establishment of a low shear quiescent microgravity like cell culture environment. As such, the rigid walls dampen all propagation waves, inhibiting both random hydrodynamic flow and gas mass transfer; now disclosed in the present invention.
All the current horizontal rotating cell culture systems developed by NASA strive to achieve a quiescent microgravity like environment, having origins in the NASA space program. There are over 100 scientific articles and abstracts demonstrating or suggesting microgravity induced aberrancy in cell growth, cell differentiation, cell movement, cell size and shape, cell physiology or general human physiology, occurring in their rotating culture systems or in actual microgravity. The normal mammalian hydrodynamic milieu differs from a quiescent microgravity like environment. In a natural environment, cells are constantly moved, pulled or forced to settled in different directions depending on the position of the organism, ie; standing, sitting or lying down. Movements from walking, running, and breathing agitate the cells from random directions, thereby adding shear and random hydrodynamic flow. An intrauterine environment is characterized by cells developing in amniotic fluid with random hydrodynamic flow, high mass transfer and defined by a compliant elastic uterus. The present invention more closely emulates the intrauterine hydrodynamic and gas mass transfer environment where positional changes, breathing, and movement continuously disrupt boundary layers and randomizes the direction of hydrodynamic flow. More specifically, the present invention relates to a novel hydrodynamic and gas exchange environment that suspends cells and facilitates 3-dimensional tissue growth.